Devices and Methods Related to Airway Inflammation

ABSTRACT

This disclosure relates to devices, assays, and methods related to airway inflammation caused by polymorphonuclear neutrophils (PMNs). In certain embodiments, the disclosure relates to a model device that emulates the changes in airway cell physiology due to transmigration of PMNs from blood to the cells at the air-liquid interface. In certain embodiments, the airway cells are supported on a collagen layer wherein the collagen layer is further supported by a porous polymer from which PMNs can migrate. In certain embodiments, the disclosure contemplates adding bacteria, fungi and/or viruses to the device to emulate disease states. In certain embodiments, the disclosure relates to the use of the model system to test compounds to identify drug candidates and diagnose subjects with airway-related diseases and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International ApplicationNumber PCT/US2014/052048 filed Aug. 21, 2014, which claims priority toU.S. Provisional Application No. 61/868,722 filed Aug. 22, 2013. Theentirety of each of these applications is hereby incorporated byreference for all purposes.

BACKGROUND

Cystic Fibrosis (CF) is a life threatening recessive condition caused bymutations in the gene coding for the CF Transmembrane conductanceRegulator (CFTR) protein. CFTR is expressed chiefly by exocrineepithelia, and its dysfunction in CF patients leads to abnormal ionbalance, hydration, pH, and redox properties of exocrine secretionsleading to organ disease. Hallmarks of CF airway disease includebronchiectasis, inflammation by polymorphonuclear neutrophils (PMNs)from blood, obstruction by mucus, and infection by bacteria. Airwaydisease is the main cause of morbidity in CF, starting after birth andprogressing at a different pace depending on patients, leading to lungfailure. Thus, there is a need to identify improved treatments.

The CFTR protein is a channel for small anions and a regulator ofvarious other transport mechanisms that is expressed chiefly in exocrineepithelia. In the airways, defective CFTR function hampers thereabsorption of luminal glucose and amino acids by the epithelium,driving microbial growth and auxotrophic adaptation. The CF airwayepithelium also secretes high levels of pro-inflammatory mediators, evenin the absence of overt infection, leading to the recruitment of bloodPMNs. Recruited PMNs produce reactive oxygen species (ROS) and releasegranule enzymes, e.g., myeloperoxidase (MPO), neutrophil elastase (NE),and matrix metalloprotease-9 (MMP-9) which lead to local production ofhighly active ROS, tissue proteolysis, high luminal amino acid levels,and changes in epithelial cells and glands. NE itself is a highly activeprotease and can reprogram epithelial cells and glands. Reflecting onits multiple pathological activities, NE activity in the airway fluidhas been identified as a strong predictor of declining CF lung function.

Tirouvanziam et al. report functional and signaling changes in viableinflammatory neutrophils homing to cystic fibrosis airways. Proc NatlAcad Sci USA, 2008, 105(11):4335-9. Makam et al. report activation ofcritical, host-induced, metabolic and stress pathways marks neutrophilentry into cystic fibrosis lungs. Proc Natl Acad Sci USA, 2009,106(14):5779-83. Laval et al. report metabolic adaptation of neutrophilsin cystic fibrosis airways involves distinct shifts in nutrienttransporter expression. J Immunol, 2013, 190(12):6043-50.

Hibbert et al. report a method of transporting epithelial cellmonolayers. US Application Publication 2010-0047907

Karp et al. report an in vitro model of differentiated human airwayepithelia cells. Methods Mol Med, 2002, 188:115-137. Fulcher et al.report human airway epithelial cell cultures. Methods Mol Med, 2005,107:183-206.

SUMMARY

This disclosure relates to devices, assays, and methods related toairway inflammation caused by polymorphonuclear neutrophils (PMNs). Incertain embodiments, the disclosure relates to a model device thatemulates the changes in airway cell physiology due to transmigration ofPMNs from blood to the apical aspect of the cells grown at air-liquidinterface (ALI). In certain embodiments, the airway cells are supportedon a collagen layer wherein the collagen layer is further supported by aporous polymer from which PMNs can migrate. In certain embodiments, thedisclosure relates to the use of the model system to test compounds toidentify drug candidates and diagnose subjects with airway-relateddiseases and conditions.

In certain embodiments, the disclosure relates to devices used toevaluate airway cells. Typically the devices comprise: a layer ofcollagen between a first compartment and a second compartment; cells onthe layer of collagen inside of the first compartment, wherein the cellsare airway cells; and a porous layer next to the layer of collageninside the second compartment, configured with a pore size sufficientfor the transmigration of PMNs through the collagen layer, wherein thesecond compartment comprises a sample comprising PMNs configured tocontact the porous layer.

In certain embodiments, the disclosure relates to methods comprising,contacting a solution with the layer of airway cells contained indevices as reported herein wherein said solution induces the PMNs in thesecond compartment to migrate to the first compartment; collecting asample from the first compartment comprising transmigrated PMNs; andanalyzing the transmigrated PMNs for a physical property.

In certain embodiments, the disclosure relates to methods of drugscreening by testing a compound for treating PMN-mediated airwayinflammation by adding a test compound into the first and/or secondcompartment(s) and identifying changes to the physical properties of thetransmigrated PMNs compared to a simulated normal and/or diseased stateor condition.

In certain embodiments, the disclosure relates to methods of predicting,aiding or assisting in the diagnoses, determining the risk of,monitoring the progression, or identifying candidate agents fortreatment of a subject with an airway-related disease or condition.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D show data on PMN hyperexocytosis in chronic and early CF.A/B: Analysis during acute pulmonary exacerbations (APE) and at steadystate show increased surface CD63 and decreased surface CD16 in CFairway vs. blood PMNs. C: Airway PMNs in chronic CF patients, but nothealthy controls (HC), are predominantly CD63hiCD16lo (designated as A2subset). D: In CF infants, the A2 subset of airway PMNs is very highcompared to disease controls (DC). * and ** for P<0.05 and 0.01.

FIGS. 2A-B show data on caspase-1 and pinocytic activities in CF airwayPMNs. A: Analysis during acute pulmonary exacerbation (APE) and steadystate (SS) show increased caspase-1 activity in live CF airway vs. bloodPMNs, based on a cell-permeable probe binding the active site of theenzyme. B: Airway PMNs in chronic CF (steady-state) show higherpinocytosis than blood PMNs, as measured by Lucifer Yellow uptake. * and** indicate P<0.05 and 0.01, respectively.

FIGS. 3A-C illustrate an in vitro model recapitulating CF small airwayPMN inflammation. A: illustrates primary granule release by airway PMNsin chronic CF disease which release NE and MPO as part of areprogramming process that is characterized by the physical propertiesof high surface levels of CD63 (hyperexocytosis), low surface levels ofCD16 (reflecting loss of phagocytic receptor expression), highintracellular activity of caspase-1, and high pinocytic activity. B:illustrates a model of PMN-induced airway inflammation based on an ALIculture of H441 human small airway cells, with CF airway fluid used asthe apical medium to induce the transmigration and reprogramming ofnaïve blood PMNs placed on the basal side. C: illustrates that collagen(star) is on the Alvetex® scaffold which simulates the natural path forPMN transepithelial migration from the basal side to the apical side dueto CF airway fluid or chemoattractants.

FIG. 4 shows data indicating CF airway fluid supernatant (ASN) is morepotent that standard chemoattractants in recruiting PMNs in ourtransmigration model. Data normalized to the CF ASN condition. Note thatthe differential between CF ASN and fMLF, IL-8 and LTB4 increases withtime due to increased survival in ASN.

FIGS. 5A-C show data indicating CF airway fluid supernatant (ASN)induces PMN reprogramming in the model system. A: CD63 (exocytosis) andcaspase-1 (pro-inflammatory activity) are induced and CD16 (phagocyticreceptor) decreased in PMNs transmigrated (TM) into CF ASN. B:Chemoattractants do not induce PMN reprogramming, as judged by CD63(exocytosis) and Lucifer Yellow (pinocytosis). C: Exocytosis andpinocytosis are correlated during PMN reprogramming by CF ASN in vitro.

FIG. 6 shows data indicating transcriptional profiling of CF airwayPMNs. Competitive hybridization microarray of blood and airway PMNs frompatients reveals marked changes in transcription induced by recruitmentto CF airways. Unsupervised clustering of Fluidigm data distinguishedairway PMNs (right most cluster) from blood PMNs and monocytes.

FIGS. 7A-B show an embodied device of the disclosure in the form of aplate array—FIG. 7A is a side view and the FIG. 7B is a top view. Thedevice comprises a housing (3) configured to form a first compartment(1) and a second compartment (2), wherein a layer of collagen (4) isbetween the first compartment (1) and the second compartment (2). Airwaycells (5) are on the layer of collagen inside of the first compartment(1). A porous layer (6) is next to/below the layer of collagen insidethe second compartment (2). The porous material is configured with apore size sufficient for the migration of polymorphonuclear neutrophils(7) through the collagen layer. PMNs break down the collagen duringtransmigration. The device contains a plurality of wells, enablingmultiplexed screening experiments (8).

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, physiology, and the like, which arewithin the skill of the art. Such techniques are explained fully in theliterature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

A “subject” refers to a human, newborn, fetus, laboratory animal, ordomestic pet.

As used herein, methods for “aiding diagnosis” or “assisting indiagnosis” both refer to methods that assist in making a clinicaldetermination regarding the presence or progression of the disease orconditions, and may or may not be conclusive with respect to thedefinitive diagnosis.

As used herein, the term “predicting” refers to making a finding withnotably enhanced likelihood of developing the disease or condition.

As used herein, the term “fluid lung sample” or “lung fluid” and thelike, refer to a biological sample derived from or around the lung ortrachea. Examples include bronchoalveolar lavage fluid and airwayepithelial cell fluid. Examples of obtaining a fluid lung sample includeby dilution of bronchial brushings, mucosal biopsies, thoracentesis,lavage, sputum induction, or spontaneous expectoration. Inbronchoalveolar lavage, a solution is injected onto airway epithelialcells, e.g., in the lung, and suctioned out to collect cells lining theairways.

As used herein, “blood sample” encompasses a biological sample which isderived from blood obtained from an individual and can be used in adiagnostic or monitoring assay. The definition encompasses blood,plasma, and serum.

As used herein, a “reference value” can be an absolute value; a relativevalue; an average value; a median value, a mean value, or a value ascompared to a particular control or baseline value. A reference valuecan be based on an individual sample or a large number of samples, suchas from patients or normal individuals.

A “normalized measured” value refers to a measurement taken and adjustedto take background into consideration. Background subtraction to obtaintotal fluorescence is considered a normalized measurement. Thebackground subtraction allows for the correction of backgroundfluorescence that is inherent in the optical system and assay buffers.

The term “binding agent” refers to a molecule, preferably aproteinaceous molecule, that specific binds a target with a greateraffinity than other molecules, e.g., is at least 10 times as great, butoptionally 50 times as great, 100, 250, or 500 times as great, or evenat least 1000 times as great as the affinity of another antibody,peptide, or polypeptide of a random sequence of similarly size andoverall hydrophobicity. Typically the specific binding agent is anantibody, such as a polyclonal or monoclonal antibody (mAb), singlechain antibody, or fragments; however, other protein and non-proteinbased binging agents are contemplated such as, affibody molecules,affilins, affitins, anticalins, avimers, DARPins, fynomers, Kunitzdomain peptides, monobodies, known or unknown, naturally occurring orsynthetic ligands, receptors, or fragments thereof. Identifying abinding agent is routine to a skilled artisan, e.g., by screening atarget molecule in a phage display, yeast display, bacterial display,ribosome display, or mRNA display library. See Miersch et al, Methods,2012, 57(4):486-98. Screening small molecule libraries produced bycombinatorial solid phase synthesis is also a method for identifyingbinding agents. See Made et al., Beilstein J Org Chem, 2014,10:1197-212.

Devices and Methods of Use

In certain embodiments, the disclosure relates to devices used toevaluate airway cells. Typically the devices comprise a layer ofcollagen between a first compartment and a second compartment; cells onthe layer of collagen inside of the first compartment, wherein the cellsare airway cells; and a porous layer next to the layer of collageninside the second compartment, configured with a pore size sufficientfor the migration of PMNs through the collagen layer, wherein the secondcompartment comprises a sample comprising PMNs configured to contact theporous layer.

Typically, the layer of cells is airway cells, however, other cell typessuch as intestinal epithelial cells, stem cells or pluripotent cells arecontemplated. In certain embodiments, the airway cells are human ornon-human diseased or normal cells such as cystic fibrosis (CF) airwaycells, non-CF bronchiectasis airway fluid, chronic obstructive pulmonarydisease (COPD) airway cells, asthmatic airway cells, diabetic airwaycells, bronchial smooth muscle cells, lung microvascular endothelialcells, bronchial tracheal epithelial cells, lung fibroblasts, pulmonaryartery cells, small airway epithelial cells.

In certain embodiments, the disclosure relates to a device having ahousing configured to form a first compartment and a second compartment;a layer of collagen between the first compartment and the secondcompartment; cells on the layer of collagen inside of the firstcompartment; and a porous layer next to the layer of collagen inside thesecond compartment, configured with a pore size sufficient for themigration of PMNs through the collagen layer. Typically, the secondcompartment comprises a sample comprising PMNs configured to contact theporous layer such as a blood sample.

In certain embodiments, the collagen layer is reconstituted from apowder that was the result of extraction from a natural source such asdemineralized bond, tail, tendon, of rats, cows, humans or otheranimals. In one embodiment, the collagen layer is reconstituted from anacid extracted human placenta type I collagen.

In certain embodiments the porous layer is a biocompatible or inertpolymer. In certain embodiments the porous layer contains polystyrene.The pores may be created by drilling holes through the polystyrenepolymer or by the creation of a polymer matrix. In certain embodiments,the porous layer is Alvetex® which is a polystyrene scaffold typicallywith a thickness of about 200 microns. It has varying pore sizes, themajority about 36-40 microns. In certain embodiments the porous layerhas a thickness between 150-250 microns. In certain embodiments, theporous layer comprises pores between 5 and 50 microns.

In certain embodiments, the first compartment comprises lung fluidobtained from a subject diagnosed, at risk of, exhibiting symptoms, orsuspected of having an airway disease or condition wherein the lungfluid is configured to contact the airway cells in the device to inducetransmigration of PMNs in the second compartment.

In certain embodiments, the device is arranged in a well of a substratewherein said substrate houses a plurality of wells each comprising thefirst and second compartments described above. The number of wells canbe between 5 and 100, or 96, or 2 and 1000, or more. In certainembodiments, the substrate housing is glass, metal, plastic, orcombinations thereof.

As used herein, “collagen” refers to a material containing collagenfibril aggregates made up of three polypeptide strands in a helix,sometimes called tropocollagen. There is a predominant sequence patternof three amino acids, two of which are predominately glycine and proline(or hydroxypoline) wherein the third may be any of various other aminoacid residues. Glycine typically accounts for about a third of thesequence. Proline or hydroxyproline typically accounts for about onesixth of the total sequence. Differences in the primary structure (aminoacid sequence) establish differences between the different types ofcollagen, e.g., collagen I, II, III, IV, etc. In the natural biochemicalprocesses of forming collagen, hydroxylation of lysine and proline aminoacids sometimes occurs which also allows for their glycosylated. Type Icollagen is a natural component of skin, bone, tendon, placenta, andother fibrous connective tissues.

Collagen may be extracted from natural products in the presence ofprotein degrading enzymes such as pepsin and reconstituted. A thresholdnumber of lysine residues in collagen allow for water solubility inacidic conditions varying on the acidity of the solution and the extentof lysine substitution. Acidic extraction followed by dehydration, e.g.,lyophilization, typically results in a collagen powder. For example, seeNiyibizi et al., J. Biol. Chem., 259:14170 (1984) for a process ofobtaining collagen from human placenta. The acid soluble collagen powdercan be dissolved in water with acetic acid to form gels of collagen.Dehydration of the gels and be configured to form a collagen layer,sheet, or film. In certain embodiments, a layer of collagenreconstituted from acid soluble extraction is applied on the porouslayers.

In certain embodiments, the disclosure contemplates the use ofreconstituted collagen that is dissolved in an aqueous acidic acidsolution and applied directly on a porous polystyrene material underconditions to form a gel or film. In certain embodiments, the collagenis type I collage extracted from human placenta; however, other forms ofcollagen are contemplated. Reconstituted collagen sheets typically areable to absorb fluid.

Crosslinking agents may be used to form a covalent bridge between lysineresidues within collagen. By introducing lysine into a protein structureon can added additional proteins by molecular crosslinking to collagen.

In certain embodiments, the disclosure relates to methods comprising,contacting a solution with the layer of airway cells contained indevices as reported herein wherein said solution induces the PMN cellsin the second compartment to migrate to the first compartment;collecting a sample from the first compartment comprising transmigratedPMNs; and analyzing the transmigrated PMNs for a physical property.

In certain embodiments, the solution is an airway fluid is obtained froma subject diagnosed, at risk of, exhibiting symptoms, or suspected ofhaving an airway disease or condition. In certain embodiments, solutionis airway fluid obtained from a subject diagnosed with, CF, non-CFbronchiectasis, COPD, or asthma.

In certain embodiments, adding bacteria, fungi and/or viruses to thefirst and/or second compartment are done to allow modelling alongvarious paths to emulate different disease states.

In certain embodiments, alternative sources of the collagen supportedcells in the first compartment and PMNs are contemplated. In certainembodiments, the cells are airway epithelium, small airway cells (frombronchioles) a genetically engineered H441 cell line with a specificknock-out (e.g., of the cftr or beta-enac gene) and/or knock-in (e.g.,expression of a specific cftr mutant) gene, other Clara cell lines(human or other species), primary bronchiolar epithelial cells fromcommercial or academic sources, large airway cells (from trachea andbronchi), matched primary cells expressing wild-type and KO or mutantcftr (e.g., 16HBE and CFBE, NuLi and CuFi lines (U of Iowa), primarybronchial epithelial cells from commercial or academic sources. Incertain embodiments, the cells are intestinal epithelium such as Caco-2and T84 cell lines with specific knock-out and/or knock-in genes.

In certain embodiments, the airway fluid is from subjects with chronicairway diseases such as non-CF bronchiectasis, COPD, asthma (bothallergic and non-allergic asthma have a predominance of PMNs in thelamina propria and lumen, having also eosinophils represented, from lungtransplant patients). CF being the major indication for lungtransplantation, this source of fluid allows one to look at the impactof the in vivo replacement of CF lungs by normal lungs on the PMNreprogramming properties of the airway fluid. In certain embodiments,the airway fluid is from conditioned media from airway or PMN culturesor conditioned media from viral, bacterial or fungal cultures, with orwithout live organisms.

In certain embodiments, the PMNs are from human subjects with variousconditions such as airway diseases, e.g., CF, non-CF bronchiectasis,COPD, asthma or subjects with primary immunodeficiencies, e.g., chronicgranulomatous disease (CGD), Familial mediterranean fever (FMF), alpha-1antitrypsin deficiency (AlAT).

In certain embodiments, the PMNs are from other species wild-type andknockout and/or knock-in mice, as a means to dissect out pathways key toPMN responsiveness to cues present in the fluid or Rhesus PMNs, as ameans to document evolutionary changes in PMN responsiveness incomparison with human and mouse PMNs, or innate cell subsets such aseosinophils, which play an effector role in asthma and macrophages,which play a regulatory role in CF, non-CF bronchiectasis, COPD, andasthma.

In certain embodiments, analyzing the transmigrated PMN cells for aphysical property is mixing the transmigrated PMNs with an antibody,binding agent, or other molecular probe that upon binding and detectionindicates cell viability, apoptosis, pinocytosis, pro-inflammatoryactivity, or oxidative activity.

In certain embodiments, the physical property is the expression ofsurface markers on the surface of the transmigrated PMN cells, orexpression of a polypeptide, enzyme, or nucleic acid within thetransmigrated PMNs.

In certain embodiments, the physical property is internalization of afluorescent molecule, such as Luciferase Yellow, into the transmigratedPMNs. In certain embodiments, analyzing the transmigrated PMN cells fora physical property is mixing the transmigrated PMN cells with LuciferYellow.

In certain embodiments, analyzing the transmigrated PMN cells for aphysical property is mixing the transmigrated PMN cells with an antibodyor other binding agent that binds to a cell surface marker wherein theantibody or binding agent is conjugated to a fluorescent molecule or theantibody or binding agent is made fluorescent thereafter, and separatingantibody or binding agent bound transmigrated PMN cells by flowcytometry.

In certain embodiments, the surface marker is selected from CD45, CD3,CD14, CD16, CD33, CD63 and CD66b or combinations thereof.

In certain embodiments, analyzing the transmigrated PMN cells for aphysical property is mixing the transmigrated PMN cells with a molecularprobe that binds with active caspase-1. See Smolewski et al., Detectionof caspase activation by fluorochrome-labeled inhibitors: multiparameteranalysis by laser scanning cytometry. Cytometry 44:73-82 (2001). Incertain embodiments, the probe is a fluorochrome-labeled inhibitor ofcaspase activation (FLICA). FLICA ligands are carboxyfluorescein(FAM)-labeled peptide fluoromethyl ketones (FMK). FMK moiety interactswith the cysteine of the active center to form a thiomethyl ketone andirreversibly inactivates the enzyme. The specificity of binding isprovided by the sequence of amino acids in the tetrapeptide (e.g., VEID)moiety. The fluorescent tag (carboxyfluorescein, FAM) is located on theother end of the FLICA molecule.

Flow cytometry is a laser-based technique that may be employed incounting, sorting, and detecting cells by suspending particles in astream of fluid and passing them by an electronic detection apparatus. Aflow cytometer has the ability to discriminate different particles onthe basis of color. Differential dyeing of particles with differentdyes, emitting in two or more different wavelengths allows the particleto be distinguished. Multiplexed analysis allows one to perform multiplediscrete assays in a single tube with the same sample at the same time.

In one example, particles may be beads each with distinctivecombinations of fluorophores that confer each bead a specified, uniquecolor code. Beads act as a solid surface that is coated with captureantibodies of interest. The bead can be mixed with an epitope, e.g.,cell surface marker, providing a bead that is conjugated through anantibody to a cell of interest. Additional detection antibodies(fluorescent or made fluorescent for a reporter signal) can create amulticolored bead-cell complex. The beads are passed through a flowcell, on a laser instrument that utilizes two-laser system, in which onelaser detects the color code of each bead, and the second laser detectsthe reporter signal, hence cells concentration.

In certain embodiments, the particles may be polystyrene microspheresthat bear carboxylate functional groups on the surface. The particlescan be covalently coupled to amine-containing ligands or antibodies to asurface marker through surface carboxylate groups; alternatively,avidin-coupled particles can be used for binding biotinylated ligands orantibodies. The bound cell can be exposed to fluorescent antibodies ornucleic acid detection reagents to provide a specific signal for eachreaction in a multiplexed assay. Each fluorescent detection reagentbinds specifically to a cell surface marker that is present on only onebead set in a multiplexed assay. Fluorescent molecules may be labeledwith a green-emitting fluorophore such as Bodipy® (Molecular Probes) orfluorescein isothiocyanate.

In certain embodiments, the disclosure contemplates individual sets ofparticles of fluorescently coded particles conjugated with ligands orantibody to PMN surface markers. After mixing the particles with asample, the particles are mixed with fluorescent detection antibodies orany fluorescent molecule that will bind to the surface markers. Mixturesof particles containing various amounts of fluorescence on theirsurfaces are analyzed with a flow cytometer. Data acquisition, analysis,and reporting are performed on the particles sets. As each particle isanalyzed by the flow cytometer, the particle is classified into itsdistinct set on the basis fluorescence and values are recorded.

The data generated by flow cytometers can be plotted in a singledimension, to produce a histogram, or multi-dimensional plots, e.g.,when multiple fluorescent moieties are attached or contained within thesame cells. For example, it is specifically contemplated that antibodiesto CD63 and CD16 and other probes disclosed herein can be conjugatedwith or made fluorescent with differing fluorescent moieties allowingthe multi-dimension evaluation of PMN cells by flow cytometry. Theregions on these plots can be sequentially separated, based onfluorescence intensity, by creating a series of subset extractions,termed “gates.” For example, antibodies to CD45, CD3, CD14, CD16, CD33,CD63 and CD66b for can be used for gating of PMNs obtained from bloodand lung fluid samples (BALF), e.g.,

CD45^(lo)CD33^(hi)CD66b^(hi)CD63^(lo)CD16^(hi) as A1 subset;

CD45^(lo)CD33^(hi)CD66b^(hi)CD63^(hi)CD16^(lo) as A2 subset;

CD45^(hi)CD33^(hi)CD66b⁻ as monocytes/macrophages;

CD45^(hi)CD3⁺ as T-cells;

CD45^(hi)CD33^(hi)CD66b^(hi)CD16⁻) as eosinophils;

CD45^(hi)CD66b⁻CD16⁺ as NK cells; and

CD45^(hi)CD3⁻CD33⁻CD66b⁻CD16⁻ as B-cells.

Non-antibody based probes can be used for gating viability (Live/Dead),apoptosis (Annexin V), pinocytosis (Lucifer Yellow uptake),pro-inflammatory activity (caspase-1) and oxidative activity (ROS).Multiple functional outcomes can be measured simultaneously on blood andlung fluid subsets. It is also contemplated that PMNs can be mixed withfluorescent bacteria to assess their phagocytic ability.

Another contemplated test setup for determining physical properties ofPMN cells disclosed herein include the use of an immune assay, aradioimmunoassay, or a ligand binding assay, e.g., enzyme-linkedimmunosorbent assay. The cell is immobilized on a solid support such asa polystyrene microtiter plate either non-specifically by adsorption tospots or zones on the surface or specifically by capture by aligand—molecule that has affinity for the cell, e.g., antibody specificto the cell surface maker. After the biomarker is immobilized presenceof the cells is detected. In one example, a detection antibody (e.g.,second antibody) is mixed with the surface. If the biomarker is in thespot, the detection antibody may form a complex with the cell. Thedetection antibody may be covalently linked to an enzyme that creates asignal upon exposure to appropriate conditions, e.g., by adding anenzymatic substrate to produce a visible signal which indicates thequantity of antigen in the sample. The detection antibody may be itselfdetected or monitored by a variety of techniques, such as through anantibody with affinity for the detection antibody conjugated to anenzyme. Typically the surface is washed to remove any cells that are notspecifically bound.

In certain embodiments, the cells can be immobilized on the surface byligand binding and a detection reagent will bind specifically to a cellssurface marker. The detection reagent may be conjugated to an enzyme togenerate a signal that can be quantified. For example, Rica & Stevensreport an enzyme label that controls the growth of gold nanoparticlesand generates colored solutions with distinct tonality when the analyteis present. See Nature Nanotechnology, 7:821-824 (2012).

In certain embodiments, the cells are captured with a ligand or antibodyon a surface and the cells is labeled with an enzyme. In one example, adetection antibody conjugated to biotin or streptavidin—to create abiotin-streptavidin linkage to on an enzyme that contains biotin orstreptavidin. A signal is generated by the conversion of the enzymesubstrate into a colored molecule and the intensity of the color of thesolution is quantified by measuring the absorbance with a light sensor.Contemplated assays may utilize chromogenic reporters and substratesthat produce some kind of observable color change to indicate thepresence of the cell. Fluorogenic, electrochemiluminescent, andreal-time PCR reporters are also contemplated to create quantifiablesignals.

Drug Screening

In certain embodiments, the disclosure relates to methods of drugscreening by testing a compound for treating PMN airway inflammation byadding a test compound into the first or second compartments andidentifying changes to the physical properties of the transmigrated PMNscompared to a simulated normal and/or diseased state or condition.

In certain embodiments, the disclosure relates to methods of testing acompound for treating PMN-associated airway inflammation comprising,contacting a solution with the layer of airway cells contained in adevice of as reported herein, wherein said solution is capable ofinducing the PMNs in the second compartment to migrate to the firstcompartment providing transmigrated PMNs, and said solution is capableof inducing the transmigrated PMNs to increase release of NE and MPO,increase caspase-1, increase expression of CD63 and decreased expressionof CD16 on said cells; contacting a test compound with the layer ofairway cells; collecting a sample from the first compartment comprisingtransmigrated PMNs; and analyzing the transmigrated PMNs to determinewhether the test compound reduced the ability of the solution to inducethe transmigrated PMNs to increase release of NE and MPO, increasecaspase-1, increase expression of CD63, or decreased expression of CD16on said cells.

In certain embodiments, the disclosure relates to methods of testing acompound for treating PMN-associated airway inflammation comprising,contacting a solution with the layer of airway cells contained in adevice as reported herein, wherein said solution is capable of inducingthe PMNs in the second compartment to migrate to the first compartmentproviding transmigrated PMNs, and said solution is capable of inducingthe transmigrated PMNs to increase release of NE and MPO, increasecaspase-1, increase expression of CD63 and decreased expression of CD16on said cells; contacting a test compound with the PMNs in the secondcompartment; collecting a sample from the first compartment comprisingtransmigrated PMNs; and analyzing the transmigrated PMNs to determinewhether the test compound reduced the ability of the solution to inducethe transmigrated PMN cells to increase release of NE and MPO, increasecaspase-1, increase expression of CD63 or decreased expression of CD16on said cells.

In certain embodiments, the disclosure relates to methods of testing acompound for treating PMN-associated airway inflammation comprising,contacting a solution with the layer of airway cells contained in adevice as reported herein, wherein said solution is capable of inducingthe PMNs in the second compartment to migrate to the first compartmentproviding transmigrated PMNs, and said solution is capable of inducingthe transmigrated PMNs and cause decay, death, or reduced functioning ofthe airway cells; contacting a test compound with the layer of airwaycells; and analyzing the airway cells to determine whether the testcompound reduced the ability of the solution to cause decay, death, orreduced functioning of the airway cells.

In certain embodiments, the disclosure relates to methods of testing acompound for treating PMN airway inflammation comprising, contacting asolution with the layer of airway cells contained in a device asreported herein, wherein said solution is capable of inducing the PMNsin the second compartment to migrate to the first compartment providingtransmigrated PMNs, and said solution is capable of inducing thetransmigrated PMNs and cause decay, death, or reduced functioning of theairway cells; contacting a test compound with the PMNs in the secondcompartment; and analyzing the airway cells to determine whether thetest compound reduced the ability of the solution to cause decay, death,or reduced functioning of the airway cells.

Diagnostic Methods

In certain embodiments, the disclosure relates to methods of predicting,aiding or assisting in the diagnoses, determining the risk of,monitoring the progression, or identifying candidate agents fortreatment of a subject with an airway disease or condition. In certainembodiments, the disclosure provides methods for monitoring progressionof an airway disease or conditions by evaluation of PMNs subject withdevices and methods disclosed herein providing measured levels of PMNpopulations or markers, e.g., CD63, CD16 and other surface proteins,caspase-1 and other intracellular enzymes, nucleic acids and makingcomparisons to reference levels or previous test results. In the case ofmonitoring disease progression one, two, or more samples, may beanalyzed daily, weekly, monthly, bimonthly, biannually, or annually. Incertain embodiments, the subject has been diagnosed with an airwaydisease, e.g., the subject has been diagnosed with CF due to abnormalsweat chloride levels, raised blood concentrations of immunoreactivetrypsinogen or mutated copies of the cftr gene.

In certain embodiments, the disclosure relates to methods comprising:obtaining an airway fluid sample from a subject; contacting the airwayfluid sample with the layer of airway cells contained in a device asreported herein wherein said airway fluid sample is capable of inducingthe PMNs in the second compartment to migrate to the first compartmentproviding transmigrated PMNs, determining whether the transmigrated PMNsindicate an airway disease state.

In certain embodiments, determining is performed by contacting a sampleof the transmigrated PMNs with antibody to CD63 or CD16 and detectingwhether binding occurs between the transmigrated PMNs and the antibodyto CD63 or CD16 using flow cytometry; diagnosing the patient as havingan airway disease if there is an increase expression of CD63 ordecreased expression of CD16 on said cells compared to normal PMNs ordiagnosing the patient as not having an airway disease if there is anexpression of CD63 or expression of CD16 on said cells that is similarto normal PMNs.

In certain embodiments, determining is performed by contacting a sampleof the transmigrated PMNs with cell permeable probe that binds activecaspase-1 and detecting whether binding occurs between caspase-1 in thetransmigrated PMNs and the cell permeable probe using flow cytometry;diagnosing the patient as having an airway disease if there is anincreased/altered expression of active caspase-1 compared to normal PMNsor diagnosing the patient as not having an airway disease if there is anexpression of active caspase-1 in said cells that is similar to normalPMNs.

In certain embodiments, determining is performed by contacting a sampleof the transmigrated PMNs with a fluorescent moiety that indicatespinocytic activity and detecting whether the fluorescent moiety isinternalized in the transmigrated PMNs using flow cytometry; diagnosingthe patient as having an airway disease if there is an increaseinternalization of the fluorescent moiety compared to normal PMNs ordiagnosing the patient as not having an airway disease if there is aninternalization of the fluorescent moiety in said cells that is similarto normal PMNs.

A physical property, e.g., surface marker expression, profile isconsidered “identified” as being useful for aiding in the diagnosis,diagnosis, stratification, monitoring, and/or prediction of airwaydisease when it is significantly different between the subsets ofsamples tested. Levels are “significantly different” when theprobability that the property has been identified by chance is less thana predetermined value. The method of calculating such probability willdepend on the exact method utilizes to compare the levels between thesubsets. As will be understood by those in the art, the predeterminedvalue will vary depending on the number the number of samples utilized.Accordingly, predetermined value may range from as high as 50% to as lowas 20, 10, 5, 3, 2, or 1%.

The process of comparing a measured value and a reference value can becarried out in any convenient manner appropriate to the type of measuredvalue and reference value for a physical property at issue. Measuringcan be performed using quantitative or qualitative measurementtechniques, and the mode of comparing a measured value and a referencevalue can vary depending on the measurement technology employed. Forexample, when a qualitative colorimetric assay is used to measuresurface marker levels, the levels may be compared by visually comparingthe intensity of the colored reaction product, or by comparing data fromdensitometric or spectrometric measurements of the colored reactionproduct (e.g., comparing numerical data or graphical data, such as barcharts, derived from the measuring device). As with qualitativemeasurements, the comparison can be made by inspecting the numericaldata, by inspecting representations of the data (e.g., inspectinggraphical representations such as bar or line graphs).

Examples CF Airway PMNs

Hyperexocytosis of primary granules occurs in chronic and early CFairway disease. Using direct analysis of airway PMNs by cytometry,experiments indicate that NE and MPO release in CF stems in large partfrom hyperexocytosis by live PMNs. Data indicates that CF airway PMNsare characterized by high surface levels of CD63 (hyperexocytosis, FIG.1A) and low surface levels of CD16 (reflecting loss of phagocyticreceptor expression, FIG. 1B), such that live airway PMNs in chronic CFdisease are primarily of the CD63^(hi)CD16^(lo) phenotype (designated as“A2” subset), in contrast with the major CD63^(lo)CD16^(hi) subset (“A1”subset) found in healthy control airways (FIG. 1C). Data also indicatesthat CF children (N=9, 3-40 months, with very mild disease and withoutany chronic infection) also showed a very high frequency of A2 PMNs inthe airways (FIG. 1D). This suggests that PMN hyperexocytosis occursearly in CF pathogenesis.

CF airway PMNs show high pinocytic and caspase-1 activities. Usingcytometry-based phenotyping, airway PMNs in chronic CF induce twoactivities, besides hyperexocytosis. First, they show high intracellularactivity of caspase-1 (FIG. 2A), a non-apoptotic caspase that producesthe pro-inflammatory mediator interleukin-1β (IL-1β). Consistently, highIL-1β levels in CF airway fluid were found. Second, CF airway PMNs showhigh pinocytic activity, based on Lucifer Yellow (LY) uptake (FIG. 2B).

In Vitro Model Recapitulating the Pathological Process of CF Airway PMNReprogramming

Research on CF airway disease is often focused on large, gland-bearingairways (bronchi), which are a major site of disease. However, earlysigns of CF airway disease occur in the small airways (bronchioles).While multiple animal and in vitro models exist for large airways, thosefor small airways are rare. Disclosed herein is a setup forrecapitulating PMN transmigration into small airways (FIG. 3). Sincetransmigration is a process in PMN conditioning, a unique air-interfacesmall airway PMN transmigration model has been designed that phenocopiespro-exocytosis and pro-survival changes seen in vivo. This modelintegrates Clara-like cells, representative of the small airwaysbelieved to be affected during CF disease. Features in this model areindependently controllable, including apical fluids, PMNs added to thebasal side, and drugs added on either or both sides, which provideunique opportunities for mechanistic studies of the process ofPMN-driven airway inflammation. This model provides the ability to studymechanisms and screen potential drug modulators of PMN-driven smallairway inflammation, for example in CF. This platform has severalelements: 1) a well-differentiated human small airway epithelial barriermaintained at air-liquid interface; 2); a porous support that emulatesthe structure of the small airway interstitium; 3) naive human bloodPMNs, added to the basal side of the model; and 4) apical stimuli(bronchoalveolar lavage fluid (BALF), airway fluid rid of cells andbacteria, chemoattractants), added on top to recruit PMNs through theepithelial layer and into the lumen. When airway fluid from chronic CFpatients is added apically, naive blood PMNs from healthy controls (orCF patients) are recruited into the lumen and develop strikingly similarfeatures to those of CF airway PMNs in vivo. This model can be used totest a sample, e.g., BALF, from a subject suspected of having an airwaylung disease for the ability to induce changes on PMNs. Chronic CFairway fluid alone (rid of cells and bacteria) can trigger PMNrecruitment and reprogramming in this model. Chemoattractants used inlieu of CF airway fluid lead to lower recruitment and do not inducereprogramming. See FIGS. 4 and 5.

This model can also be used to test and assess drugs targeted at PMNreprogramming and on their ability to alter inflammation. Whileconventional HTS strategies focus on molecular entities, a strategy isto focus on this disease process (PMN transmigration and reprogramming),integrating target cells (PMNs) and the relevant milieu (CF airwayfluid), and enabling drug testing in in vivo-like conditions.

In a typical experiment, the apical side will be bathed with CF airwayfluid (positive control for transmigration and reprogramming),chemo-attractants (positive control for transmigration and negativecontrol for reprogramming), or medium (negative control for both).Molecule for testing can be added to the basal side to test for systemicactivity (by oral or intravenous administration) or can be combined withCF airway fluid to test for administrating through the lungs, e.g.,administration by inhalation.

Analyses of PMNs collected in the model can be accomplished by usingmultiplexed flow cytometry, such that multiple endpoints of alterationof activity can be assessed simultaneously with a multi-color stainingcombination (compatible with automated plate cytometers). For live PMNgating testing may be based on scatter, Live/Dead negativity, andpositivity for CD66b. For live PMN enumeration testing may be basedabsolute count using spiked counting beads. For exocytosis testing maybe based surface CD63. For caspase-1 activity testing may be basedintracellular staining with caspase-1 active site probe. For pinocytosistesting may be based on Lucifer Yellow uptake. With this integratedanalytical scheme, viability and number of transmigrated PMN and keyphenotypes linked to reprogramming can be assessed in a flow cytometryassay. In addition to this set of cell-based endpoints, confirmatoryassays may be used to measure cell and fluid properties using imagecytometry (to visualize pinocytic vesicles), Luminex (to measuresecretion of caspase-1 derived cytokines), spectrometry (to measure NEand MPO release by exocytosis, and Lucifer Yellow decrease due touptake), Western Blot (to measure cleavage and thereby activation ofintracellular caspase-1) and transcriptomics (to assess transcriptionalreprogramming in PMNs).

This model can be used for finding inhibitors of PMN-driven small airwayinflammation in CF and other PMN-driven inflammation in other diseasesand conditions, including, but not limited to non-CF bronchiectasis,chronic obstructive pulmonary disease (COPD) and neutrophilic asthma.

CF Airway PMNs are Metabolically and Transcriptionally Altered

Airway PMNs in chronic CF trigger anabolic signaling along the mammaliantarget of rapamycin (mTOR) pathway and increase glucose, amino acid andphosphate transporter expression. To assess if metabolic changes wereassociated with transcriptional changes, sorted airway and blood PMNswere analyzed by microarrays Changes in mRNA expression over 2-fold wereseen in ˜10% of genes. Gene Ontology analysis shows that: 1)downregulated mRNAs in CF airway PMNs relate to wound healing (p<10-13),programmed cell death (p<10-6), and phosphatase activity (p<10-2); and2) upregulated mRNAs relate to translational elongation (p<10-94),ribosome formation (p<10-83) and cellular component biogenesis(p<10-10). Thus, PMNs inhibit death pathways and induce anabolicpathways while being activated in CF airways. To gain further insightinto this question, the multiplexed qPCR Fluidigm platform was used as aprofiling method as shown in FIG. 6. A chip was designed with 48transcripts involved in signaling (mTOR, and other pathways relevant toCF airway PMNs), functional responses, and a chosen set of highlyup/down-regulated mRNAs, per the microarray data. The mRNA data suggestthat PMNs undergo key transcriptional changes in the CF airway lumen.

1. A device comprising, a layer of collagen between a first compartmentand a second compartment; cells on the layer of collagen inside of thefirst compartment, wherein the cells are airway cells; and a porouslayer next to the layer of collagen inside the second compartment,configured with a pore size sufficient for the migration ofpolymorphonuclear neutrophils (PMNs) to the collagen layer, wherein thesecond compartment comprises a sample comprising PMNs configured tocontact the porous layer.
 2. The device of claim 1, wherein the sampleis blood.
 3. The device of claim 1, wherein the first compartmentcomprises a solution of chemo-attractants or lung fluid configured tocontact the airway cells.
 4. The device of claim 1, wherein the first orsecond compartment comprises bacteria, fungi and/or viruses.
 5. A methodcomprising, ing a solution with the layer of airway cells contained in adevice of claim 1 wherein said solution induces the PMNs in the secondcompartment to migrate to the first compartment; collecting a samplefrom the first compartment comprising transmigrated PMNs; and analyzingthe transmigrated PMNs for a physical property.
 6. The method of claim5, wherein the solution is an airway fluid is obtained from a subjectdiagnosed, at risk of, exhibiting symptoms, or suspected of having anairway-related disease or condition.
 7. The method of claim 5, whereinsolution is airway fluid obtained from a subject diagnosed with cysticfibrosis, non-CF bronchiectasis, COPD, or asthma.
 8. The method of claim5, wherein analyzing the transmigrated PMNs for a physical property ismixing the transmigrated PMNs with an antibody that binds to a cellsurface marker wherein the antibody is conjugated to a fluorescentmolecule or the antibody is made fluorescent thereafter, and separatingantibody bound transmigrated PMNs by flow cytometry.
 9. The method ofclaim 6, wherein the surface markers which is selected from CD45, CD3,CD14, CD16, CD33, CD63 and CD66b, or combinations thereof.
 10. A methodof testing a compound for treating PMN-associated airway inflammationcomprising, ing a solution with the layer of airway cells contained in adevice of claim 1, wherein said solution is capable of inducing the PMNsin the second compartment to migrate to the first compartment providingtransmigrated PMNs, and said solution is capable of inducing thetransmigrated PMNs to increase release of NE and MPO, increasecaspase-1, increase expression of CD63 and decreased expression of CD16on said cells; contacting a test compound with the layer of airwaycells; collecting a sample from the first compartment comprisingtransmigrated PMNs; and analyzing the transmigrated PMNs to determinewhether the test compound reduced the ability of the solution to inducethe transmigrated PMNs to increase release of NE and MPO, increasecaspase-1, increase expression of CD63, or decreased expression of CD16on said cells.
 11. A method of testing a compound for treatingPMN-associated airway inflammation comprising, ing a solution with thelayer of airway cells contained in a device of claim 1, wherein saidsolution is capable of inducing the PMN cells in the second compartmentto migrate to the first compartment providing transmigrated PMNs, andsaid solution is capable of inducing the transmigrated PMNs to increaserelease of NE and MPO, increase caspase-1, increase expression of CD63and decreased expression of CD16 on said cells; contacting a testcompound with the PMN cells in the second compartment; collecting asample from the first compartment comprising transmigrated PMN cells;and analyzing the transmigrated PMNs to determine whether the testcompound reduced the ability of the solution to induce the transmigratedPMNs to increase release of NE and MPO, increase caspase-1, increaseexpression of CD63 or decreased expression of CD16 on said cells.
 12. Amethod of testing a compound for treating PMN-associated airwayinflammation comprising, ing a solution with the layer of airway cellscontained in a device of claim 1, wherein said solution is capable ofinducing the PMNs in the second compartment to migrate to the firstcompartment providing transmigrated PMNs, and said solution is capableof inducing the transmigrated PMNs and cause decay, death, or reducedfunctioning of the airway cells; contacting a test compound with thelayer of airway cells; and analyzing the airway cells to determinewhether the test compound reduced the ability of the solution to causedecay, death, or reduced functioning of the airway cells.
 13. A methodof testing a compound for treating PMN-associated airway inflammationcomprising, ing a solution with the layer of airway cells contained in adevice of claim 1, wherein said solution is capable of inducing the PMNsin the second compartment to migrate to the first compartment providingtransmigrated PMNs, and said solution is capable of inducing thetransmigrated PMNs and cause decay, death, or reduced functioning of theairway cells; contacting a test compound with the PMNs in the secondcompartment; and analyzing the airway cells to determine whether thetest compound reduced the ability of the solution to cause decay, death,or reduced functioning of the airway cells.
 14. A method for determiningwhether a human patient has an airway disease, comprising: obtaining anairway fluid sample from a subject; ing the airway fluid sample with thelayer of airway cells contained in a device of claim 1, wherein saidairway fluid sample is capable of inducing the PMNs in the secondcompartment to migrate to the first compartment providing transmigratedPMNs; and determining whether the transmigrated PMNs indicate an airwaydisease.
 15. The method of claim 14, wherein said determining isperformed by contacting a sample of the transmigrated PMNs with antibodyto CD63 or CD16 and detecting whether binding occurs between thetransmigrated PMNs and the antibody to CD63 or CD16 using flowcytometry; diagnosing the patient as having an airway disease if thereis an increase expression of CD63 or decreased expression of CD16 onsaid cells compared to normal PMNs or diagnosing the patient as nothaving an airway disease if there is an expression of CD63 or expressionof CD16 on said cells that is similar to normal PMNs.
 16. The method ofclaim 14, wherein said determining is performed by contacting a sampleof the transmigrated PMNs with a cell-permeable probe that bindscaspase-1 and detecting whether binding occurs between caspase-1 in thetransmigrated PMNs and the cell-permeable probe using flow cytometry;diagnosing the patient as having an airway disease if there is analtered expression of active caspase-1 compared to normal PMNs ordiagnosing the patient as not having an airway disease if there is anexpression of active caspase-1 in said cells that is similar to normalPMN cells.
 17. The method of claim 14, wherein said determining isperformed by contacting a sample of the transmigrated PMNs with afluorescent moiety that indicates pinocytic activity and detectingwhether the fluorescent moiety is internalized in the transmigrated PMNsusing flow cytometry; diagnosing the patient as having an airway diseaseif there is an increase internalization of the fluorescent moietycompared to normal PMNs or diagnosing the patient as not having anairway disease if there is an internalization of the fluorescent moietyin said cells that is similar to normal PMNs.